1. Field of the Invention
The present invention relates to a method for synthesizing biologically active bis-heterocyclic compounds, e.g. bis-indoles. In particular, the present invention provides methods for making Soritin compounds such as Soritin B, bis-(1H-indol-3-yl)-acetic acid methyl ester, Soritin C, bis-2,2-(1-methyl-indol-3-yl) acetic acid methyl ester, Soritin D, bis-2,2-(1-methyl-indol-3-yl) acetic acid.
2. Description of the Related Art
Bis-heterocyclic compounds, such as bis-indoles, have been previously described as having antimicrobial, antitumor or antiviral activity. See U.S. Pat. Nos. 5,955,462; 6,090,811; and 6,291,501, which are herein incorporated by reference. Specifically, the bis-indole compounds known as topsentins are disclosed in U.S. Pat. No. 4,866,084 and nortopsentins are disclosed in U.S. Pat. No. 4,970,226, which are herein incorporated by reference. Dragmacidin and its related compounds isolated from the marine sponge of the Dragmacidon sp. are disclosed in U.S. Pat. No. 4,895,844, which is herein incorporated by reference. These patents are herein incorporated by reference. These compounds as well as the homocarbonyl topsentins and hamacanthins have also been described as having inhibitory activity against cellular inflammatory responses. See U.S. Pat. Nos. 5,290,777 and 5,464,835, which are also hereby incorporated by reference.
U.S. Pat. Nos. 6,444,697 and 6,323,233 and U.S. patent application No. 20010056112 disclose bis-heterocyclic compounds known as Soritin compounds having the following formula:
wherein R1-10 are the same or different selected from —H, —OH, halogen, —COOH, —COOR, C1–C8 alkyl, C1–C8 alkoxyl, mesyl, tosyl, —OCOR, or NZ1Z2 (wherein the Zs can be the same or different);
X1 and X2 are the same or different selected from —H, —R, —COY, C(NZ1)Y;
Y is —H, —OH, NZ1Z2 (wherein the Z1 and Z2 can be the same or different) C1–C8 alkyl, C1–C8 alkoxyl or an amino acid linked through the amine functionality forming an amide bond;
Z1 and Z2 are the same or different and independently selected from —H, —OH, C1–C8 alkyl, C1–C8 alkoxyl or —COR; and
R is C1–C8 alkyl, or aryl.
The prior art patents and publications disclose methods for making a few Soritin compounds via synthetic methods that require prohibitive reaction conditions, indirect synthetic routes, and low yields. For example, Hogan and Sainsbury describe a process for making Soritin B, methyl bis(3-indolyl)-acetate which requires a temperature of −20° C. See SYNTHESIS (1984) 10:872. Likewise Amat-Guerri et al. describe a process similar to Hogan and Sainsbury which provides low yields of the desired Soritin compounds. See CHEM. LETT. (1981) 4:511–541. Earle et al. disclose a process for making Soritin C by reacting methyl chloromethoxyacetate with indole in the presence of zinc chloride at 0° C. for 8 hours. See TETRAHEDRON LETT. (1991) 32(43):6171–6174.
U.S. patent application Ser. No. 10/211,370, filed 5 Aug. 2002, discloses methods for making Soritin B and C, wherein the route of synthesis must pass through Soritin A or Soritin D. Specifically, Soritin B is made according to the following method: One-tenth (0.1) mol of indole was suspended in 500 ml of distilled water in a 1000 ml round bottom flask fitted with a reflux condenser. One-tenth (0.1) mol of glyoxylic acid (50% solution in water, Aldrich) was added to the flask. The flask was covered with foil to protect the reaction products from light. The reaction mixture was stirred and slowly heated to 50° C. using an oil bath. Once 50° C. was reached, 20 ml of 1M H2SO4 was added to the reaction mixture. The reaction mixture was heated to about 85° C. (to melt the indole) and the reaction allowed to proceed to completion (>95% conversion of indole to Soritin A). Typically, the reaction is complete within 2 hours. The reaction progress can be monitored by TLC using silica gel plates eluted with heptane-ethyl acetate 3:1 (v/v). Compounds can be visualized either by charring after treatment with 2% vanillin in H2SO4, or by UV absorbance. The product precipitates from solution as a light pink solid. The product was removed by filtration and then washed with cold distilled water to yield substantially pure Soritin A. The product was freeze-dried for 24 hours to remove water. The final yield was 0.06 mol of Soritin A.
The freeze dried Soritin A was then dissolved in 500 ml of dry methanol in a round bottom flask fitted with a reflux condenser and protected from light. Six (6) drops of concentrated H2SO4 were added to the reaction mixture which was heated to reflux for about 6 hours. The reaction progress was monitored by TLC using silica gel plates eluted with heptane-ethyl acetate 3:1 (v/v). Compounds were visualized either by charring after treatment with 2% vanillin in H2SO4, or by UV absorbance. Once the reaction was judged complete (typical conversion after 6 hours is about 60%), the solvent was reduced in volume by about two-thirds (⅔) by distillation under reduced pressure. Saturated aqueous NaCl solution (500 ml) was added. The aqueous solution was extracted three times with ethyl acetate (200 ml portions). The combined ethyl acetate extracts were then washed two times with saturated sodium bicarbonate solution (100 ml) and then dried over magnesium sulfate. The dried ethyl acetate solution was filtered to remove the MgSO4, and then treated with activated carbon (100 mesh) to remove colored impurities. The filtrate was then concentrated by distillation under reduced pressure to yield the crude reaction product. To remove unreacted starting material and indole formed by decomposition of Soritin A, the material was chromatographed over silica gel using a step gradient of ethyl acetate in heptane. Fractions are monitored by TLC as above. Fractions which were substantially pure Soritin B were combined to give an isolated yield of 0.025 mol of Soritin B.
Soritin C was made according to a similar process as follows: One-tenth (0.1) mol of N-methylindole was suspended in 500 ml of distilled water in a 1000 ml round bottom flask fitted with a reflux condenser. One-tenth (0.1) mol of glyoxylic acid (50% solution in water, Aldrich) was added to the flask. The flask was covered with foil to protect the reaction products from light. The reaction mixture was stirred and slowly heated to 50° C. using an oil bath. Once 50° C. was reached, 20 ml of 1M H2SO4 was added to the reaction mixture. The reaction mixture was heated to about 80° C. and the reaction allowed to proceed to completion (>95% conversion of N-methyl indole to Soritin D). Typically the reaction is complete within 2 hours. The reaction progress can be monitored by TLC using silica gel plates eluted with heptane-ethyl acetate 3:1 (v/v). Compounds can be visualized either by charring after treatment with 2% vanillin in H2SO4, or by UV absorbance. The product precipitates from solution as a light tan solid. The product was removed by filtration and then washed with cold distilled water to yield substantially pure Soritin D. The product was freeze-dried for 24 hours to remove water. The final yield was 0.05 mol of Soritin D (VI).
The freeze dried Soritin D was then dissolved in 500 ml of dry methanol in a round bottom flask fitted with a reflux condenser and protected from light. Six (6) drops of concentrated H2SO4 were added to the reaction mixture which was heated to reflux for about 6 hours. The reaction progress was monitored by TLC using silica gel plates eluted with heptane-ethyl acetate 3:1 (v/v). Compounds were visualized either by charring after treatment with 2% vanillin in H2SO4, or by UV absorbance. Once the reaction was judged complete (typical conversion after 6 hours is about 60%), the solvent was reduced in volume by about two-thirds (⅔) by distillation under reduced pressure. Saturated aqueous NaCl solution (500 ml) was added. The aqueous solution was extracted three times with ethyl acetate (200 ml portions). The combined ethyl acetate extracts were then washed two times with saturated sodium bicarbonate solution (100 ml) and then dried over magnesium sulfate. The dried ethyl acetate solution was filtered to remove the MgSO4, and then treated with activated carbon (100 mesh) to remove colored impurities. The filtrate is then concentrated by distillation under reduced pressure to yield the crude reaction product. To remove unreacted starting material, material was chromatographed over silica gel using a step gradient of ethyl acetate in heptane. Fractions were monitored by TLC as above. Fractions which were substantially pure Soritin C were combined.
Thus, the prior art methods of making Soritin compounds are indirect, i.e. have numerous process steps, and require unduly reaction conditions. Additionally, the intermediates of the prior art indirect methods breakdown, i.e. degrade, easily, thereby reducing yields and increasing degradation products or impurities. Therefore, a need still exists for faster, more efficient, easier, and higher yielding methods for making Soritin compounds and Soritin compositions having increased purity.